The proposed research is a biochemical-genetic study of mit rRNA splicing in Neurospora. Recent studies have shown that the gene that codes for the 25 S (large subunit) mitochondrial rRNA of Neurospora contains an intervening sequence of 3000 bp which is not present in mature 25 S RNA (Kuntzel, 1978). We have found that this intervening sequence is excised in events leading to the synthesis of the normal 32 S pre-rRNA. We have also identified two temperature-sensitive, nuclear mutants which, at the non-permissive temperature, accumulate pre-rRNAs containing most or all of the intervening sequence. Specific aims are: (a) to continue to characterize physiological defects in the mutants, (b) to characterize the structure of the large RNAs, (c) to use the large RNAs as substrate to isolate the RNA splicing enzymes and (d) to isolate and characterize revertants of the mutants. These studies are intended to provide insight into the mechanism and physiological role of RNA splicing which should be relevant to all eukaryotic cells.